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Shed Antigen-Induced Blocking Effect on CAR-T Cells Targeting Glypican-3

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Shed Antigen-Induced Blocking Effect on CAR-T Cells Targeting Glypican-3 Open access Original research J Immunother Cancer: first published as 10.1136/jitc-2020-001875 on 8 April 2021. Downloaded from Shed antigen-induced blocking effect on CAR- T cells targeting Glypican-3 in Hepatocellular Carcinoma Luan Sun,1 Fang Gao,1 Zhanhui Gao,1,2 Lei Ao,1 Na Li,1 Sujuan Ma,1 Meng Jia,3,4 Nan Li,5 Peihua Lu,6 Beicheng Sun,7 Mitchell Ho,5 Shaochang Jia,4 Tong Ding,1 1 Wei Gao To cite: Sun L, Gao F, Gao Z, ABSTRACT including sorafenib and lenvatinib; most et al. Shed antigen- induced Background Glypican-3 (GPC3), a cell surface HCC patients cannot receive curative treat- blocking effect on CAR- T glycoprotein that is pathologically highly expressed in cells targeting Glypican-3 in ment based on current restrictive criteria and hepatocellular carcinoma (HCC), is an attractive target for 2 3 Hepatocellular Carcinoma. exhibit an extremely poor prognosis. Addi- immunotherapies, including chimeric antigen receptor Journal for ImmunoTherapy tionally, immunotherapies using immune (CAR) T cells. The serum GPC3 is frequently elevated in of Cancer 2021;9:e001875. checkpoint inhibitors have shown benefits doi:10.1136/jitc-2020-001875 HCC patients due to the shedding effect of cell surface GPC3. The shed GPC3 (sGPC3) is reported to block the in only a small proportion of HCC patients, LS and FG contributed equally. function of cell-surface GPC3 as a negative regulator. possibly because of the complex immunosup- 4 5 Accepted 09 March 2021 Therefore, it would be worth investigating the potential pressive microenvironment. Thus, there is influence of antigen shedding in anti-GPC3 CAR-T therapy an urgent need to develop new effective ther- for HCC. apeutic strategies for HCC. Methods In this study, we constructed two types of CAR-T Glypican-3 (GPC3) is a cell- surface glyco- cells targeting distinct epitopes of GPC3 to examine how phosphatidylinositol (GPI)- anchored protein sGPC3 influences the activation and cytotoxicity of CAR-T that belongs to the heparan sulfate (HS) cells in vitro and in vivo by introducing sGPC3 positive proteoglycan family, which plays important patient serum or recombinant sGPC3 proteins into HCC cells or by using sGPC3-overexpressing HCC cell lines. roles in cell growth, differentiation and migra- Results Both humanized YP7 CAR-T cells and 32A9 tion. Many studies have shown that GPC3 is CAR-T cells showed GPC3-specific antitumor functions highly expressed in HCC, while its expression in vitro and in vivo. The existence of sGPC3 significantly is absent in most nonmalignant adult tissues. http://jitc.bmj.com/ inhibited the release of cytokines and the cytotoxicity of GPC3 is currently used as an informative anti-GPC3 CAR-T cells in vitro. In animal models, mice immunohistochemical biomarker for HCC, carrying Hep3B xenograft tumors expressing sGPC3 and it is believed to be an attractive target exhibited a worse response to the treatment with CAR- T for HCC therapy.6 7 Various GPC3- targeted cells under both a low and high tumor burden. sGPC3 strategies have been developed or evaluated bound to CAR-T cells but failed to induce the effective 8 9 activation of CAR-T cells. Therefore, sGPC3 acted as in HCC. Our previous work showed that on September 30, 2021 by guest. Protected copyright. dominant negative regulators when competed with cell GPC3- specific immunotoxin and antibody surface GPC3 to bind anti-GPC3 CAR-T cells, leading to an drug conjugates presented potent anticancer 10 11 inhibitory effect on CAR-T cells in HCC. activity in vitro and in vivo. Recently, Conclusions We provide a proof-of- concept study chimeric antigen receptor (CAR) T cells, © Author(s) (or their demonstrating that GPC3 shedding might cause worse which have shown promising curative effects employer(s)) 2021. Re- use response to CAR-T cell treatment by competing with cell in hematological tumors, have been applied permitted under CC BY-NC. No surface GPC3 for CAR- T cell binding, which revealed a new for the development of novel GPC3-targeted commercial re- use. See rights mechanism of tumor immune escape in HCC, providing therapies in HCC and have shown prelimi- and permissions. Published by a novel biomarker for patient enrolment in future clinical BMJ. nary effects in some studies12–15 and clinical trials and/or treatments with GPC3-targeted CAR-T cells. 8 9 16 For numbered affiliations see trials. However, the response rate in the end of article. clinical setting is still far from satisfactory, and it is not clear how HCC cells may escape from Correspondence to BACKGROUND Dr Wei Gao; gao@ njmu. edu. cn Liver cancer is a leading cause of cancer- CAR- T cells. related death with an increasing incidence GPC3 is composed of a 70 kDa core protein Dr Tong Ding; worldwide. Primary hepatocellular carcinoma and two HS side chains with highly negative dingtong@ njmu. edu. cn (HCC) is the most common type of liver charges, which can help GPC3 recruit and 1 Dr Shaochang Jia; cancer. HCC is generally resistant to chemo- concentrate many essential ligands from the shaochangjiabio@ 163. com therapy, radiotherapy and specific inhibitors, tumor microenvironment and facilitate their Sun L, et al. J Immunother Cancer 2021;9:e001875. doi:10.1136/jitc-2020-001875 1 Open access J Immunother Cancer: first published as 10.1136/jitc-2020-001875 on 8 April 2021. Downloaded from recognition of corresponding receptors.17–19 Our previous donors’ blood, obtained under the ethics protocol studies have demonstrated that GPC3 functions as a core- approved by Nanjing Jinling Hospital (Nanjing, Jiangsu). ceptor to modulate Wnt/β-catenin signaling to promote The isolated PBMCs were cultured in RPMI 1640 Medium cell proliferation in HCC.19–21 Similar to other glypicans, (Roswell Park Memorial Institute 1640 Medium, HyClone, GPC3 can be released from the cell surface and can be Logan, Utah, USA) supplemented with 10% fetal bovine found around tumors or in the circulation.22 Several serum (Gibco, Murphysboro, IL), 100 U/mL penicillin, studies have shown that serum GPC3 levels are signifi- and 0.1 mg/mL streptomycin, with incubation under 5% cantly elevated in HCC patients compared with healthy CO2 at 37°C. All other cell lines were cultured in Dulbec- individuals23–25 and that local tumor GPC3 concentrations co’s modified Eagle’s medium (HyClone) supplemented in HCC may also be much higher than those in normal with 10% fetal bovine serum (VACCA, Murphysboro, Illi- tissues. Previous studies also showed that recombinant nois, USA), 100 U/mL penicillin and 0.1 mg/mL strep- soluble GPC3 could inhibit HCC cell growth in vitro tomycin (HyClone). All cell lines were confirmed free of and in vivo,26 27 suggesting that shed GPC3 (sGPC3) may mycoplasma contamination by PCR. compete with cell- surface GPC3 to bind GPC3- interacting molecules or may even block GPC3- targeted therapies. Antibodies and recombinant proteins Many tumor antigens are actively shed from cell YP7 and 32A9 are GPC3- specific monoclonal antibodies 30–32 surface, and such shedding can be expected to signifi- generated in our previous studies. YP7 is a mouse cantly influence the efficiency of anticancer treatment antibody and was humanized (named hYP7) for the targeting these antigens.28 29 However, it is not very clear generation of CAR- T cells. The recombinant sGPC3 how antigen shedding is involved in CAR-T therapy, espe- (Q25- S550) protein with a hexa- histidine tag at the cially in solid tumors, which compared with hematolog- C- terminal end, or with a wild- type human Fc (hFc) tag ical malignancies exhibit a relatively isolated and enclosed or mutant hFc tag containing three mutations (N297A/ microenvironment. Therefore, it is worth analyzing the L234A/L235A) reported to abolish unexpected binding 33 34 role of sGPC3 in CAR-T cell therapy regimens for HCC, to the Fcγ receptor were cloned into the pFUSE with the expectation of identifying novel diagnostic and/ vector (InvivoGen, San Diego, California, USA). All the or prognostic indications. above plasmids were expressed in HEK293T cells. After In the current study, based on GPC3-specific antibodies collecting the supernatant, protein purification was 35 developed in our previous work, we established GPC3- accomplished following our previous protocol. Recom- targeted CAR- T cells and studied how sGPC3 influenced binant sCD19(P20- K291) with either Fc tag or mutant Fc CAR- T cell activation and cytotoxicity in HCC models in tag were also purified as the negative control antigen. vitro and in vivo. In short, our results showed that sGPC3 Serum GPC3 detection can impair the antitumor activities of CAR-T cells in HCC A 32A9 IgG (4 µg/mL) was used to coat ELISA wells at by competing with cell- surface GPC3, resulting in the 4°C overnight. After blocking, diluted serum or standard escape of tumors from immunotherapy. http://jitc.bmj.com/ recombinant GPC3 protein were added and incubated at 4°C overnight. After washing, biotin labeled YP7-IgG (1 µg/mL) was added and then detected by Streptavi- METHODS din- HRP antibody (Life Tech, Peoria, Illinois, USA). TMB Clinical samples and cells and H SO were added to detect the OD value. The HCC tumor tissue, normal liver tissue specimens 2 4 450nm and serum used in the current study were obtained from Generation and expansion of CAR-T cells on September 30, 2021 by guest. Protected copyright. patients recruited from Nanjing Drum Tower Hospital hYP7, 32A9 and FMC63-based second- generation 4- 1BB (Nanjing, Jiangsu, China) between January 2018 and CARs were designed and packaged into a lentivirus as we April 2019, and Nanjing Jinling Hospital (Nanjing, described previously.32 Then, T cells were isolated from Jiangsu, China) between October 2020 and December PBMCs using a CD8+CD4+ negative isolation kit (STEM- 2020.
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